Abstract

Aneuploidy, an abnormal number of chromosomes, has previously been considered irremediable. Here, we report findings that euploid cells increased among cultured aneuploid cells after exposure to the protein ZSCAN4, encoded by a mammalian-specific gene that is ordinarily expressed in preimplantation embryos and occasionally in stem cells. For footprint-free delivery of ZSCAN4 to cells, we developed ZSCAN4 synthetic mRNAs and Sendai virus vectors that encode human ZSCAN4. Applying the ZSCAN4 biologics to established cultures of mouse embryonic stem cells, most of which had become aneuploid and polyploid, dramatically increased the number of euploid cells within a few days. We then tested the biologics on non-immortalized primary human fibroblast cells derived from four individuals with Down syndrome—the most frequent autosomal trisomy of chromosome 21. Within weeks after ZSCAN4 application to the cells in culture, fluorescent in situ hybridization with a chromosome 21-specific probe detected the emergence of up to 24% of cells with only two rather than three copies. High-resolution G-banded chromosomes further showed up to 40% of cells with a normal karyotype. These findings were confirmed by whole-exome sequencing. Similar results were obtained for cells with the trisomy 18 of Edwards syndrome. Thus a direct, efficient correction of aneuploidy in human fibroblast cells seems possible in vitro using human ZSCAN4.

Analyses of three additional human trisomy 21 primary fibroblast cells with Syn-mRNAs. The fraction (%) of cells with two dots. The results of five biological replications for AG05397 cells and three biological replications for AG06872 and AG08942 cells are shown for each condition. For each sample, the numbers of nuclei examined to score two or three dots by FISH are shown above bars (n). GFP, cells treated with Syn-GFP; hZ4, cells treated with Syn-hZSCAN4. P, one-tailed t-test. P*, one-tailed Welch's t-test.

Analyses of human trisomy 21 primary fibroblast cells with SeV vectors. (A) Schematic overview of an experimental approach using SeV vectors. (B) The fraction (%) of AG05397 and AG05024 cells with two dots (mean ± SEM). The results of three biological replications are shown for each condition. For each sample, more than 200 nuclei were examined to score two or three dots by FISH. No, non-treated control; AG, cells treated with SeV-AG-TS15; hZ4, cells treated with SeV-hZSCAN4-TS15. Treatment by SeV-TS15 was carried out at 35°C. The cells were kept at 35°C for 7 days, followed by incubation at 37°C for 2 weeks. P, one-tailed t-test. P*, one-tailed Welch's t-test. (C) The fraction (%) of AG05397 cells with two dots (mean ± SEM). The results of three biological replications are shown for each condition. For each sample, more than 200 nuclei were examined to score two or three dots by FISH. No, non-treated control; AG, cells treated with a SeV-AG-TS7; hZ4, cells treated with a SeV-hZSCAN4-TS7. Treatment by SeV-TS7 was carried out at 35°C. The cells were kept at 35°C for 7 days, followed by incubation at 37°C for 2 weeks. P*, one-tailed Welch's t-test. (D) The fraction (%) of AG06872 cells with two dots (mean ± SEM). The results of three biological replications are shown for each condition. For each sample, more than 200 nuclei were examined to score two or three dots by FISH. No, non-treated control; AG, cells treated with SeV-AG-TS7; hZ4, cells treated with SeV-hZSCAN4-TS7. Treatment by SeV-TS7 was carried out at 37°C. The cells were kept at 37°C for 7 days, followed by incubation at 39°C for 3 days and then further incubation at 37°C for 11 days. P*, one-tailed Welch's t-test. This figure is available in black and white in print and in colour at DNA Research online.

Analyses of the long-term effects of SeV vector treatment on human trisomy 21 primary fibroblasts. (A) Schematic overview of an experimental approach using SeV vectors for long-term cell cultures. The treatment by SeV-TS15 was carried out at 35°C. The cells were kept at 35°C for 6 days, followed by incubation at 37°C. Samples were taken every week and subjected to FISH analyses for counting the number of chromosome 21. (B) Assessment of AG expression by fluorescence microscopy and hZSCAN4 expression by immunohistochemistry using an antibody against human ZSCAN4. Day 7 after treating with SeV vector. BF, bright field. (C) The fraction (%) of cells with two dots. The numbers of nuclei examined to score two or three dots by FISH are shown above the bars (n). No, non-treated control; AG, cells treated with SeV-AG-TS15; hZ4, cells treated with SeV-hZSCAN4-TS15. (D) Examples of high-resolution G-banded karyotype analysis of AG05397 (trisomy 21) cells without treatment (No treatment), with a control SeV-AG treatment, or with SeV-hZSCAN4 treatment (upper, trisomy 21; lower, normal karyotype). (E) Summary of G-banded karyotype analysis of AG05397 fibroblast cells sampled in the sixth week.

Analyses of trisomy 21 and trisomy 18 cells treated with SeV vectors. (A) Telomere lengths of the same cells analysed in Fig. . Telomere length was measured by qPCR and presented as the (T/S) ratio (telomere to single copy gene). Results of three technical replications are shown. Error bars (SEM). No (P15), non-treated control AG05397 cells at an early passage (passage 15); No (P30, day 126), non-treated control AG05397 cells at passage 30, day 126 in culture; AG (P30, day 126), cells treated with SeV-AG-TS15, cultured at 35°C for 6 days, followed by continuous culture at 37°C for 120 days. hZ4 (P30, day 126), cells treated with SeV-hZSCAN4-TS15, cultured at 35°C for 6 days, followed by continuous culture at 37°C for 120 days. Cells were passaged every week. (B) Treatment of trisomy 18 (Edwards syndrome). Non-immortalized fibroblast cells derived from an individual with trisomy 18 (AG12614) were treated with SeV-TS7. The cells were treated with SeV-TS7 at 37°C and cultured for 6 days, followed by incubation at 39°C for 3 days, and then, at 37°C for 15 days. The number of chromosome 18 was assessed by FISH using a chromosome 18-specific probe. Results of three biological replications are shown. The fraction (%) of cells with two dots are shown (mean ± SEM). For each sample, more than 200 nuclei were examined to score two or three dots by FISH. No, non-treated control; AG, cells treated with SeV-AG-TS7; hZ4, cells treated with SeV-hZSCAN4-TS7. P, one-tailed t-test. (C) Treatment of trisomy 18 (Edwards syndrome). Non-immortalized fibroblast cells derived from an individual with trisomy 18 (AG12614) were treated with SeV-TS15. The cells were treated with SeV-TS15 at 35°C and cultured for 6 days, followed by incubation at 37°C for 2 weeks. The number of chromosome 18 was assessed by FISH using a chromosome 18-specific probe. Results of three biological replications are shown. The fraction (%) of cells with two dots are shown (mean ± SEM). For each sample, more than 200 nuclei were examined to score two or three dots by FISH. No: non-treated control. AG, cells treated with SeV-AG-TS15; hZ4, cells treated with SeV-hZSCAN4-TS15. P*, one-tailed Welch's t-test. (D) Summary diagram showing the correction of trisomy.